Structured biological samples for analysis by mass cytometry

ABSTRACT

Apparatus and methods for delivering biological samples to an ICP source of a mass cytometer are disclosed. Biological material is disposed on a plurality of discrete sites on a carrier. The plurality of discrete sites are configured to retain biological material and to release the biological material upon application of energy. The carrier is positioned in proximity to a gas conduit and upon release from the discrete sites, the biological material becomes entrained in a gas flow, which delivers discrete portions of biological material through the conduit to the ICP source for analysis by mass cytometry. The apparatus and methods can provide a continuous stream of discrete portions of biological material to a mass cytometer or mass spectrometer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application 62/108,911 filed Jan. 28, 2015 and U.S. ProvisionalPatent Application 62/098,890 filed Dec. 31, 2014, the contents of whichare incorporated herein in their entirety.

FIELD

The present disclosure relates to apparatus and methods for introducingbiological samples to a mass cytometer.

BACKGROUND

Mass cytometry is a newly developed technique for studying biologicalsamples. The technique was originally developed to study cellpopulations in which samples of interest containing various biologicalcells were “stained” with affinity probes such as antibodies that areattached to elemental tags. The amount of affinity probes of a giventype attached to each cell can be used to characterize each individualcell. The amount of affinity probe of each type is directly related tothe amount of the associated elemental tag. The amount of the elementaltagging material can be measured by passing the cell through aninductively coupled plasma (ICP) ion source of a mass cytometer. Thetechnique of “staining” biological material with affinity probes withelemental tags has recently been extended to the field of imaging ofbiological tissue. In imaging mass cytometry, a tissue of interest is“stained” with affinity probes/molecular tags and laser ablation can beused to extract the elemental tags from discrete locations (pixels) onthe tissue. Imaging mass spectrometry follows a similar method, exceptthat it relies on the detection of atoms naturally present in the samplerather than the labelling atoms in the elemental tags.

A limitation of mass cytometry is the efficiency of introducingbiological samples such as cells into the mass cytometer. Cellintroduction efficiency of commercial mass cytometers is about 30%. Thismeans that more than two-thirds of biological cells in a sample are lostbefore they can be recorded by the mass cytometer.

A further limitation of laser-ablation based imaging mass cytometry andlaser-ablation based imaging mass spectrometry is the low pixelrecording rate. The pixel recording rate is limited by the washout timeof the laser ablation cell and the connecting gas conduits. Even thefastest laser ablation cells have a washout time on the order of 30 ms.This is much slower than the intrinsic recording capabilities of themass cytometer or mass spectrometer. In a typical mass cytometer cellthroughput can be as high as 1000 cells per second; however, the spreadof the laser ablation plume as it travels through the laser ablationapparatus, i.e. washout time, limits the pixel rate to about 30 pixelsper second. Moreover, in the process of laser ablation each pixel ofmaterial is vaporized and converted into an aerosol. The aerosol plumeis then transported to the ICP source via a gas conduit. One of theproblems with this approach is that a fraction of the aerosol plume cancontaminate neighboring areas, e.g., neighboring pixels, of the tissuesample. Some fraction of the aerosol plume can also be lost duringtransport through the gas conduit connecting the laser ablation chamberto the ICP ion source. In addition, the gas dynamic spreading of theaerosol plume during transport to the ICP source further limits thewashout time. Therefore, in view of these constraints, it is desirableto avoid remote ablation method and aerosol plume formation of abiological sample and thereby improve the throughput of imaging masscytometry and imaging mass spectrometry.

SUMMARY

An ionization source, for example an ICP source, is able to evaporate,dissociate, and ionize biological material introduced directly into thesource and provide full elemental response to tagging componentsaccording to the standard function of the mass cytometer (or detectionof atoms naturally present in the sample, by a mass spectrometer, if thesample is not labelled). In other words, an ionization source, forexample an ICP source, of a mass cytometer is entirely capable ofinterrogating and ionizing biological material representing individualareas or pixels of a tissue. By creating a stream of individual pixelsrepresenting discrete areas of tissue or individual cells entrained in asuitable gas flow, each pixel can be analyzed by the mass cytometer ormass spectrometer. This approach has several advantages compared tomethods involving introduction of aerosol plumes into an ionizationsource, for example an ICP source, of a mass cytometer or massspectrometer.

Apparatuses and methods that facilitate introduction of biologicalmaterial directly into an ionization source, for example an ICP source,of a mass cytometer are provided. Apparatuses and methods utilizingpre-structured, addressable, biological samples able to introducediscrete material pixels into a gas stream and subsequently into anionization source, for example an ICP source, of a mass cytometer ormass spectrometer are disclosed.

In a first aspect, carriers are provided, comprising: a surface; aplurality of discrete sites disposed on the surface, wherein, each ofthe plurality of discrete sites is configured to retain a biologicalsample; and each of the plurality of discrete sites is configured torelease the biological sample upon application of an energy impulse. Insome embodiments, the carrier may include a sacrificial layer configuredto be targeted by a laser. The sacrificial layer, when ablated by thelaser, may eject biological material disposed on the sacrificial layerinto a gas stream. In some embodiments, a cushion layer may be providedbetween the sacrificial layer and the biological material. The cushionlayer may be configured to protect the biological material from theablation of the sacrificial layer.

In a second aspect, systems for introducing biological samples into anionization source, for example an ICP source, of a mass cytometer ormass spectrometer are provided, comprising: the carrier provided by thepresent disclosure; a conduit comprising an inlet and an outlet,wherein, the inlet is disposed in proximity to the surface of thecarrier; and the outlet is disposed in proximity to an ionizationsource, for example an ICP source; a gas flow configured to entrain abiological sample released from a discrete site and to direct thereleased biological sample through the conduit to the ionization source,for example the ICP source.

In a third aspect, methods of preparing a structured biological sampleare provided, comprising: providing the carrier provided by the presentdisclosure; flowing a solution comprising a plurality of discretebiological material over the surface of the carrier to cause thebiological material to be retained by the plurality of discrete sites;removing excess solution to provide a structured biological samplecomprising biological material retained on the plurality of discretesites.

In a fourth aspect, methods of preparing a structured biological sampleare provided, comprising: providing the carrier provided by the presentdisclosure, wherein, the surface comprises topographic featuresconfigured to cut a biological sample; and the topographic features aredisposed between the plurality of discrete sites; and applying abiological sample onto the surface of the carrier to cause thetopographic features to cut and section the biological sample, and tocause the sections of biological material to be retained by theplurality of discrete sites to provide a structured biological sample.

In a fifth aspect, methods of preparing a structured biological sampleare provided, comprising: providing the carrier provided by the presentdisclosure, applying a biological sample to the surface of the carrier;and sectioning the biological sample to remove portions of thebiological sample between the plurality of discrete sites to provide astructured biological sample.

In a sixth aspect, methods of delivering a biological sample into an ICPtorch are provided, comprising: providing the carrier provided by thepresent disclosure, wherein the plurality of discrete sites comprises abiological sample; positioning one of the plurality of discrete sites inproximity to an inlet of a conduit, wherein the conduit comprises anoutlet disposed in proximity to an ionization source, for example anionization source, for example an ICP source, of a mass cytometer ormass spectrometer; providing a gas flow over the discrete site andthrough the conduit; releasing the biological sample from the discretesite; causing the released biological sample to become entrained by thegas flow, wherein the gas flow delivers the biological sample to anionization source, for example an ICP source, of a mass cytometer ormass spectrometer; and positioning a second of the plurality of discretesites in proximity to the inlet of the conduit.

In some aspects, a method of delivering a biological sample into a masscytometry system may be provided. The method may include focusing afirst laser spot onto a film supporting the biological sample to heatthe film. The heating of the film may lift the biological sample into agas phase without complete vaporization of the biological sample. Thegas phase configured to deliver the biological sample to the masscytometry system.

The film may be coupled with a substrate. The film may be a plurality ofseparate portions of film at discrete spaced apart sites on thesubstrate. The plurality of separate portions of film may define aplurality of cell capture sites. The cell capture sites may beconfigured to capture only a single cell. A surface of gaps between theplurality of separate portions of film may be modified to repel thebiological sample.

Optionally, the first laser spot may be focused onto the film supportingthe biological sample to ablate the film. A cushion layer may bedisposed between the biological sample and the film. The cushion layermay be configured to absorb energy from the ablation of the film tolimit damage to the biological sample from the ablation of the film. Asurface of the cushion layer may be modified to preferentially capture atarget biological sample.

In some embodiments, the method may include focusing a cutting laserspot onto the film to cut around the biological sample to separate theportion of the film supporting the biological sample from a bulk of thefilm. The cutting laser spot may be from a first laser, and the firstlaser spot may be from a second laser different from the first laser.

In some aspects of the present disclosure, a method of delivering abiological sample into a mass cytometry system may be provided that mayinclude focusing a laser spot onto a film supporting the biologicalsample to ablate the film at a location adjacent to the biologicalsample. The ablation of the film at a location adjacent to thebiological sample may lift the biological sample into a gas phasewithout complete vaporization of the biological sample. The gas phasemay be configured to deliver the biological sample to the mass cytometrysystem. In some embodiments, the film may be ablated at a location thatis no more than 5 microns from the biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described below are for illustration purposes only and arenot intended to limit the scope of this disclosure.

FIG. 1 shows a schematic diagram of generic description of a masscytometer instrument configuration operating with pre-structured samplesaccording to certain embodiments.

FIG. 2 shows an example of patterned capture sites on a sample carrier.

FIG. 3 shows a liquid interface and sample carrier with patternedcapture sites on a sample carrier for introducing discrete biologicalsamples into a gas stream according to certain embodiments.

FIG. 4 shows an example of a structured tissue sample for imagingapplication and associated carrier according to certain embodiments.

FIG. 5 shows an example of a pre-structured tissue sample with gapsbetween subsections for imaging applications and associated carrieraccording to certain embodiments.

FIG. 6 shows a structured biological sample and associated carrierconfigured for laser desorption according to certain embodiments. Theproperties of light are chosen so that the carrier is virtuallytransparent while the desorption film at the capture site adsorbs thelight and causes launching of the sample portion

FIG. 7 shows a structured biological sample and associated carrierconfigured for laser desorption according to certain embodiments. Theproperties of laser light are chosen so that the light can pass throughthe sample portion with minimal damage yet cause thermal deposition atthe capture site (for instance via explosion of desorption film)

FIG. 8A to FIG. 8D shows ablation and desorption to analyze a locationof interest on a biological sample.

Reference is now made in detail to certain embodiments of devices,apparatus, and methods. The disclosed embodiments are not intended to belimiting of the claims. To the contrary, the claims are intended tocover all alternatives, modifications, and equivalents.

DETAILED DESCRIPTION

Structured biological material is used to introduce discrete samples orpixels of biological material into an ionization source, for example anICP source, of a mass cytometer or mass spectrometer. Structuredbiological material refers to material such as individual cells, tissueportions, or other aggregation of biological material, arranged indiscrete, addressable sites on a carrier. The structured biologicalmaterial can be released from the carrier and each discrete materialpixel sequentially introduced into a mass cytometer or mass spectrometerfor analysis. Benefits of sequential introduction of discrete materialpixels as opposed to random introduction of biological samples as inconventional mass cytometry or mass spectrometry include a higher sampleprocessing rate and elimination of multimers such as dimers, trimers,etc., caused by the simultaneous arrival of multiple entities. Byeliminating multimers the processing and analysis of the data can alsobe simplified.

A “pixel” refers to a portion of a material sample that is introduced atan instant in time into an ionization source, for example an ICP source,of a mass cytometer or mass spectrometer. In certain embodiments, apixel represents a cell or group of cells, and in certain embodiments, aportion of a tissue section or other discrete biological sample. A pixelmay have any suitable dimension or shape. For example, a pixel can havedimensions from 10 nm to 10 μm, from 100 nm to 10 μm, and in certainembodiments from 1 μm to 100 μm. The terms “pixel” and “discrete site”are used interchangeably. The material from a pixel is not vaporizedduring release from a carrier or during transit to an ionization source,for example an ICP source.

FIG. 1 shows a schematic diagram of a mass cytometry instrumentoperating with a structured sample. A sample carrier having discretesites or pixels containing biological material serves as the source ofbiological material. The biological material can be individually liftedfrom the discrete sites to become entrained in a carrier gas flow. Thecarrier gas flow maintains the separation of the discrete biologicalmaterial to inject the discrete biological material into an ionizationsource, for example an ICP source, where the biological material isvaporized and ionized and subsequently analyzed by mass spectrometry.

In certain embodiments, the surface of the carrier configured withdiscrete sites is mounted perpendicular to or substantiallyperpendicular to a carrier gas conduit. The inlet of the carrier gasconduit can be situated in close proximity to the surface of thecarrier. Carrier gas flowing into the gas carrier conduit can enterthrough the inlet and/or other apertures near to or included with theconduit. The conduit inlet may contain one or more skimmers or conicalshaped elements to facilitate capture of a desorbed biological sampleand/or direct desorbed sample into the gas conduit. Sometimes, thesurface of the carrier does not have discrete sites. Here, particularmethods of ablation and desorption, such as those discussed below, canbe used to analyze particular portions, such as cells or groups ofcells, of the biological sample without partitioning of the sample intodiscrete parts of the sample at discrete sites.

Various apparatus and methods for introducing an ablated plume ofmaterial into a carrier gas stream for introduction into an ionizationsource, for example an ICP source, are disclosed in InternationalApplication No. WO 2014/169394, which is incorporated by reference inits entirety. Similar apparatus and methods can be employed withembodiments provided by the present disclosure for introducing anon-vaporized sample of biological material into the carrier gas flow.The various embodiments show a carrier gas stream flowing across thesurface of the target, directed gas flows, skimmers, and gas conduitsdisposed anywhere from perpendicular to horizontal with respect to thecarrier surface.

The carrier may be mounted on a translation stage. The translation stagemay be capable of moving the surface of the carrier toward and away fromthe inlet to the gas conduit. The translation stage may also beconfigured to move the carrier in directions perpendicular to thecarrier gas conduit. The translation stage is configured to move thecarrier with respect to the inlet of the gas conduit from site to siteat a rapid rate. By moving the carrier from site to site and desorbing abiological sample at each site, a stream of discrete packets ofbiological material may be introduced into the carrier gas andtransported to an ionization source, for example an ICP source, of amass cytometer.

Sample Carrier

The invention provides a carrier comprising: a surface; a plurality ofdiscrete sites disposed on the surface, wherein each of the plurality ofdiscrete sites is configured to retain a biological sample; and each ofthe plurality of discrete sites is configured to release the biologicalsample upon application of an energy pulse.

The invention also provides a carrier comprising: a surface, wherein thesurface is configured to retain a biological sample and the surface isconfigured to release the biological sample upon application of anenergy pulse. The kinds of energy that can cause the release of thebiological sample (i.e. desorption) from the sample carrier arediscussed further below.

In some embodiments, each of the plurality of discrete sites ischaracterized by an area from 1 μm to 100 μm in circumscribed diameter.In some embodiments, the plurality of discrete sites comprises affinitymolecules configured to retain the biological sample.

In some embodiments, the carrier comprises a channel underlying theplurality of discrete sites, wherein the channel extends through athickness of the carrier; and the plurality of discrete sites comprisesa hole fluidly coupled to the channel.

In some embodiments, the plurality of discrete sites is individuallyaddressable.

As discussed in more detail below in some embodiments, the plurality ofdiscrete sites comprises a thermally desorbable material configured torelease the biological sample. Likewise, discussed in more detail below,the sample can be desorbed from the sample by thermal energy, mechanicalenergy, kinetic energy, and a combination of any of the foregoing. Oneexample is the use of laser radiation energy, in a technique calledlifting (laser induced forward transfer; see e.g. Doraiswamy et al.,2006, Applied Surface Science, 52: 4743-4747; Fernández-Pradas, 2004,Thin Solid Films 453-454: 27-30; Kyrkis et al., in Recent Advances inLaser Processing of Materials, Eds. Perriere et al., 2006, Elsivier).Accordingly, in some embodiments, the sample carrier comprises adesorption film layer. In other words, the sample carrier has a filmlayer on the surface of the carrier which is for receiving the sample,and which assists the separation of the sample from the sample carrierby absorbing laser radiation. The desorption film can absorb theradiation to cause release of the desorption film and/or the biologicalsample (e.g. in some instances the sample film desorbs from the samplecarrier together with the biological sample, in other instances, thefilm remains attached to the sample carrier, and the biological sampledesorbs from the desorption film).

In certain embodiments, a sample carrier includes a cover with a window.A cover can protect the biological samples prior to desorption and/orcan facilitate control of carrier gas flow over the sample. The carriercan be moved with respect to a window so as to bring the discretesamples on the carrier surface under the window. The window can havefeatures that facilitate capture of a biological sample by the gasconduit such as carrier gas channeling and/or electrostatic features.For example, in certain embodiments it is desirable that the carrier gasflow be laminar to facilitate the ability to systematically introducepixels of biological sample into the gas flow and subsequently into theionization source, for example an ICP torch. Use of a cover with awindow can help to maintain consistent sample-to-sample gas dynamics tofacilitate entrainment of discrete biological material pixels into thegas stream.

In certain embodiments, the sample carrier is in the form of a plate.The plate may have a substantially flat surface or other suitablesurface topography. In some embodiments, the carrier comprises atopographically defined surface configured to retain the biologicalsample. For example, in certain embodiments, the plate may have recessedfeatures for retaining biological sample and/or raised features forretaining biological sample. The surface of a plate may have channels orother suitable features for controlling gas and/or liquid flowcontaining samples across the surface of the plate and/or forfacilitating entrainment of desorbed biological sample into the carriergas flow. For example, posts may be provided with capture sites. Thepost geometry may be configured to aid in gas dynamics by allowing theflow of the carrier gas to envelope the material being lifted.

In certain embodiments, such as when the biological sample is a tissuesection, the sample carrier may be characterized by corrugated surfacefeatures having raised edges configured to cut and separate an appliedtissue section. As a tissue section is applied to a carrier surfacehaving features with cutting edges the tissue section can be separatedinto individual regions or pixels that become physically separated fromeach other.

Discrete capture sites may have a suitable area as appropriate forcapturing a particular biological material. For example, eukaryoticanimal cells have dimensions from about 10 μm to about 30 μm. A discretecapture site can include properties that facilitate the capture and/orretention of a biological material on the discrete site. The propertymay be chemical in nature such as an affinity molecule or other chemicalinteraction that facilitates adsorption or adhesion. The property may beelectrostatic, and in certain embodiments, the property may be physicalsuch as having surface features that facilitate adhesion, which caninclude roughened or specially designed adhesion surface such as havingspiny or hooked features.

In certain embodiments, a discrete site may have a multilayer structurewith layers designed for different purposes. For example, an exterior ortop layer may be configured to capture and retain a biological sample,an interior layer may be configured to heat the discrete site to desorbor release the biological material from the discrete site, and a lowerlayer may be configured for adhesion to the substrate material. In someembodiments, the top layer may provide the capture site properties thatfacilitate the capture of targeted biological material. For example, thetop layer may provide the preferential chemical binding with thebiological material (e.g., through affinity molecules or the like). Thetop layer may also provide the electrostatic forces for attractingbiological material in some embodiments and/or the surface features(e.g., roughened surface features, hooks, and/or spines or the like).

In certain embodiments, a carrier may have other geometries. Thefunctions of the carrier may include capturing a biological sample,retaining a biological sample, and/or facilitating selective release ofthe biological sample from the surface of the substrate. Thus, althoughplanar substrates or plates may be used, other structures such as beads,filaments, tubes, rods, meshes, and other suitable supporting structuresmay be employed as carriers. As appropriate for a particular carriergeometry, an associated structure may also be used to control gas flowover the surface of the carrier. For example, in certain embodiments acarrier may comprise a filament or rod within a surrounding tube. Thefilament serves as a support and the surrounding tube channels gas flowfor entraining the desorbed biological sample and for introduction intothe ionization source, for example an ICP torch.

Biological Samples

In certain embodiments, a biological sample is a cell and in certainembodiments, a tissue section.

In certain embodiments, a discrete capture site contains a single cellor multiple cells.

In certain embodiments, a biological sample can be a virus, a bacteria,a subcellular structure such as a nucleus or mitochondrion, or aeukaryotic plant cell.

Sample Capture

In certain embodiments, sample capture and release can be a dynamicprocess meaning that the capture and release processes occur more orless (e.g., nearly) simultaneously. For example, a biological samplesuch as an isolated cell may be introduced into a fluid, which flowsover the surface of a carrier. As the fluid with entrained sample flowsover discrete sites, the sample becomes captured at the discrete sites.Once a sample is captured, a desorption or lifting process may beactivated to eject the sample (e.g., a cell or the like). In someembodiments, a sample capture detector may be provided that may beoperatively coupled with the desorption or lifting mechanism (e.g.,laser, bubble-jet type mechanism) to release the captured sample intothe gas phase. The capture detector can, for example, be based onforward or side scattering information from the cell that is captured.Or, it can be based on detection of a fluorescent signal from a cellthat is stained with a particular reagent to produce the fluorescentsignal. In addition, the appearance of the cell can be detected by a CCDcamera using a microscope style observation of the capture site in someembodiments. Other—not optical methods can be employed, such as probingof solution for electrical conductivity—similar to the Coulter method orprobing solution with high frequency electrical signals. Accordingly, insome embodiments, some sites may be capturing samples while other sitesare releasing samples.

Optionally, a timing controller may be provided that is operativelycoupled with the sample capture detectors and the desorption or liftingmechanism. The timing controller may delay a release event when a cellcapture is detected until the start of the next time window in the dataacquisition setup. Accordingly, such a setup and method may convert arandom arrival of cells in a flow to an equally spaced in time releaseevents in the gas phase. This approach may reduce or otherwise eliminate“doublet” and other “multiplets” commonly detected in mass cytometrywhen the system is operating in solution mode due to the randomness ofcell arrival.

In certain embodiments, the carrier may be externally loaded withbiological sample, where the cells are fixed to the carrier prior to theintroduction of the sample carrier into the cell lifting chamber.Biological sample may be loaded passively or actively. For example, whenloaded passively a solution containing biological sample may be appliedover the surface of the carrier and the biological sample allowed toaffix to discrete sites on the carrier surface. With an actively loadedsample a biological material may be deposited at a discrete site. Thediscrete sites can have patches of binding media such that the cellswill adhere only at the discrete sites. Thus, instead of the randomlyscattered cells on the slide, the cells may be arranged on the capturesites, and optionally, the capture sites can be spaced at regularintervals to facilitate more convenient targeting for the laser. In someembodiments, the capture sites are configured or sized to hold only onecell per site.

Discrete sites may include an affinity moiety for binding tocomplementary sites of a biological sample and/or may be characterizedby other properties or chemical functionality suitable for capturinggeneric or specific biological sample. For example, a capture site caninclude molecules that enhance ionic or hydrophobic-hydrophilicinteractions with the biological material.

In certain embodiments, the region of a carrier surface not containingcapture sites may be covered with a material that cells will not adhereto and/or that causes biological material to aggregate at the discretecapture sites. An example is shown in FIG. 2 in which a “repelling”layer of material is disposed on the carrier surface not covered by thediscrete capture sites.

An example of one approach to capturing cells is disclosed by Wang etal. in which an antibody-coated (anti-EpCAM-coated) three-dimensionalnanostructured substrate that provides enhanced capture ofEpCAM-positive cells from a cell suspension. Wang et al., Angew. Chem.Int. Ed. Engl. 2009, 48(47), 8970-8973). The interdigitation ofnanoscale cellular surface components and the silicon-nano-pillar arrayenhances local topographic interactions resulting in improvedcell-capture efficiency. Other suitable apparatus and methods forcapturing cells are described in Lee et al., Nano. Lett. 2012, 12(6),2697-2704. In some embodiments, the capture sites are configured tocapture one cell per site such that single cell analysis may beperformed using mass spectrometry or mass cytometry. Optionally, in someembodiments, the capture sites may include one or more immobilizedantibodies which bind to a cell surface marker of the cell(s) ofinterest.

Mechanical methods may also be used to capture cells. FIG. 3 shows anexample of a carrier having patterned captured sites with each capturesite coupled to a channel extending through the thickness of thecarrier. Each capture site includes one or more small holes. A solutioncontaining biological material such as cells is flowed over the surfaceof the carrier and when suction is applied to a channel passing cellscan be pulled onto a capture site and retained. To release thebiological material from a capture site, pressure can be applied to thechannel to eject particles of biological material through the solutionflow and into a gas stream for injection into an ionization source, forexample an ICP source. As shown in FIG. 3, the biological material caninclude a certain amount of liquid when ejected into the gas phase. Thepressure surge can be created by a piezo actuator or by a pulsed heaterthat creates a bubble-jet effect or by a pressure spike created byabsorption of laser radiation near the biological material

The stream of gas phase particles of biological material somewhatresemble a stream from a drop on demand (DoD) droplet generator. As DoDgenerators are known to operate with an ionization source, for examplean ICP ion source, the steam of particles produced by this embodimentcould also be made compatible with the ionization source, for examplethe ICP ion source. A potential benefit of this arrangement over a DoDgenerator is that the capture sites can be designed to retain only oneparticle of biological material such as a cell. Captured biologicalmaterial can be released at equally spaced time intervals and therebyminimize confusion caused by the overlap of signal transients from eachparticle, which is a common problem when cells are entering anionization source, for example an ICP source, at random. The capturefunction of the capture site can be facilitated by simple mechanicalmeans such as obstructions in the flow or by an optional vacuum suctionchannel in which some liquid is pulled down as a cell is stopped by thediameter restriction at the capture site. Once a particle is captured itcan then be ejected into the gas flow using suitable means such as byexplosive evaporation caused by a laser pulse directed at the capturesite or by explosive evaporation caused by individually addressablemicroheaters arranged under each of the capture targets. Particles ofbiological material can also be ejected by an application of a strongelectric field. Appropriate electrode arrangements can be incorporatedon or around the capture sites for applying strong electric fields andcausing ejection of captured material. The droplets can be also beejected using microelectromechanical systems—(MEMS) basedmicroactuators. An array of MEMS micro-mirrors as used in commercialdigital projection chips represents an example of densely packed fastmoving microactuators.

In certain embodiments, pre-structuring biological tissue in only onedimension can take place on a thin ribbon or tape. The ribbon can be thearranged in a spool. The spool can be arranged in such a way that eachturn of the ribbon is separated from the next turn by a gap to avoidsmearing the sample. The ribbon can contain capture spots for samplepre-structuring. The sample can be recorded on the spool by bringingliquid sample through a capillary in contact with the moving ribbon asthe ribbon is wound on the spool. The liquid can be allowed to dry whilethe captured biological material will stay attached to the capturesites.

In certain embodiments, the carrier support is not structured and thebiological samples are irregularly distributed on the surface of thecarrier. For example, a solution of biological material such as cellscan be applied to a support. The cells can adhere randomly to thesupport. Using microscopy, the position of each of the deposited cellscan be determined and mapped. The carrier can be translated into aposition proximate a gas conduit according to the mapped position of thecells and the cells released into a carrier gas flow. The cells can beselected based on functional imaging, separation from adjacent cells,and/or other criteria. U.S. patent application Ser. No. 14/060,125, thecontents of which are incorporated herein by reference, describesmethods and systems for interrogating a sample for identifying locationsof target cells for location specific ablation that may be used withembodiments described herein. Alternatively, the sample on anon-structured carrier may be a tissue section, and similarly, regionsof interest can be identified, for example by microscopy. The regions ofinterest can be desorbed using techniques discussed herein, such ascontrolled heating at the region of interest, or by lifting (such aswhen the sample carrier comprises a desorption film layer).

Tissue

In certain embodiments, the biological material is a biological tissuesuch as a biopsy section. In these applications, one of the functions ofthe carrier is to separate the tissue into discrete areas and retain theareas prior to release. When retained by the carrier the tissue can beseparated into discrete areas having dimensions, for example, of 1 μm, 2μm, 3 μm, 4, μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, or other suitabledimension.

The tissue can be separated by means of ridges having cutting edges thatseparate the tissue when the tissue is mounted on the surface of thecarrier. An example of a carrier with serrated cutting edges betweendiscrete capture sites is shown in FIG. 4. As shown in FIG. 4, a wettissue slice is applied over a structured carrier. During applicationthe knife edges of surface features cut and separate the biologicaltissue, which further shrinks and separates during drying. The sectionedsample rests on discrete sites of the sample carrier, which areconfigured to facilitate release of the biological material from thecarrier. In certain embodiments, a wet tissue sample can be thicker thanthe height of the walls separating adjacent areas, and the walls areslightly higher or about the same height above the carrier surface asthe dried tissue slice.

Alternatively, a tissue may be mounted on the surface of a carrierhaving discrete capture sites and divided into discrete areas by cuttingthe tissue into sections using, for example, a mechanical, electrical,or laser scribe.

In certain embodiments, the substrate ridges can act to minimize thespillover of radiation used for sample desorption on the neighboringdiscrete sites or pixels. Because the dimensions of the ridges can be onthe scale of a wavelength of light, and because the light energyboundary is smeared on the scale of the wavelength the ridges that havea scale of the wavelength of light can play an important role inpreventing the light from irradiating neighboring pixels. FIG. 5 showsanother embodiment for tissue imaging applications. As shown in FIG. 5the tissue under investigation is first sectioned into individualsubsections on the sample carrier. The subsections are then sequentiallyejected into the gas phase for further analysis. Each subsection can beseparated from the remaining tissue using a variety of tools.Sub-sectioning can be used to ensure that when one subsection islaunched into the gas phase other subsections are not dragged with it.The interface between the tissue sample and the sample carrier caninclude an adhesive layer to further improve the ability of othersubsections to remain on the sample carrier. The adhesive layer can alsohave the properties of the light absorbing material suitable for aparticular wavelength.

The sectioning of each subsection from the tissue can be accomplished bymechanical tools such as blades or stamps. Alternatively, the materialbetween subsections can be removed by laser ablation in a separatesample preparation setup. In certain embodiments, the material can beremoved by a setup employing a focused electron or ion beam. The focusedelectron or ion beam can lead to particularly narrow cuts (potentiallyon the 10 nm scale) between subsections leading to a pixel size on theorder of 1 μm or in certain embodiments, 100 nm.

Other embodiments include combining the functions of the sectioningsetup in the same chamber as the ejection setup. For example, the systemcan include one laser providing light on the samples shaped to cut gapsbetween subsections by interacting and desorbing tissue material in thegaps, and then a second laser can be used to interact with lightabsorbing material under each subsection to launch the pixel ofbiological material into the gas phase. The benefit of such designs isin the sharing of a large portion of the optical path. Optionally, acushion layer may be provided between the biological material and thelight absorbing/sacrificial material. The cushion layer may be providedto protect the biological material during ablation of the lightabsorbing/sacrificial layer.

As shown in the figures the structuring of the carrier into discretesites is often periodic in nature. However, in certain applications inmay be desirable to focus analysis on one or more specific areas of atissue or a cell smear or to target individual cells from a cell sampleapplied as a thin layer. Moreover, a user may also want to obtain anoptical or other image of the tissue or a cell smear or the cellsspreads over the carrier prior to the analysis by imaging mass cytometryor imaging mass spectrometry, in order, for example, to correlate themass cytometry or mass spectrometry results with cellular ormorphological structure. Also, by taking the optical image one will beable to record an image over a large area quickly and then identify theareas of interest that require in-depth characterization by imaging masscytometry or imaging mass spectrometry. Using such methods sampling ofthe pixels or discrete sites will not necessarily be in a regularperiodic pattern but will be detailed in certain areas.

To implement strategic sample analysis, the sectioned tissue sample canbe first imaged and mapped. The mapping can represent a visual analysis.Alternatively, the mapping can be based on a distribution of a stainingmaterial, which may or may not be the same staining material as used formass cytometry analysis. For example, the staining material may be afluorophore. Alternatively, the staining material may be an element tag,which is imaged using, for example, radiography. The regions of interestfor more detailed analysis using mass cytometry or mass spectrometry canbe selected based on the mapping.

Another arrangement for imaging applications involves cutting a twodimensional sample into narrow strips (first step of pre-structuring)and then feeding the strips to a device that further cuts the strip intosubsections (second step of pre-structuring) and transferring thesesubsections into the gas phase for analysis by mass cytometer or massspectrometer. In certain embodiments, cutting a tissue sample intosubsections involves laser cutting or mechanical cutting. In certainembodiments it is desirable to make mechanical cuts by operating arotating cutting wheel with multiple cutting zones along thecircumference. For instance, a cutting wheel can rotate at a rotationalspeed of 50 revolutions per second (3000 rpm) and if the wheel itselfhas 20 equally spaced cutting sites the rate of sub-sectioning the stripwill be 1000 subsections (pixels) per second, which is a rate thatmatches the analysis throughput of a mass cytometer or massspectrometer.

In some embodiments, a sample carrier can stretch after subsections havebeen precut. This can allow one to avoid the limitation of minimal pixeldimension imposed by the diffraction of light in systems that utilizefocused laser beams for pixel ejection. Precutting with an ion beam oran electron beam can provide spatial resolution well below onemicrometer enabling imaging analysis on a sub-micrometer scale. Foldedbiological material can be used to provide the stretching function. Thisapproach can be particularly straightforward in the case of a linear(virtually one-dimensional) sample carrier. A ribbon can be folded toform an initial sample carrier. The biological sample can then beadhered to the top side of the folded ribbon and then precut at thejunctions of each fold. After that the ribbon can be unfolded to providea pre-structured biological sample with each sample pixel separated inspace by a significant distance determined by the length of each foldingstep.

In operation, it is not necessary to avoid a complete overlap of thesignals produced at the detector by material from adjacent pixels. Forexample, it can be desirable to operate the instrument with a higherpixel ejection rate such that the order of ejection of pixel subsectionsbecomes mixed during transport into the gas phase. If this operationresults in only a few out of a sequence pixels then software algorithmscan be used to restore the correct order based on interpolation to therest of the image. As can be appreciated, the apparatus and methods aresuitable for use in imaging cytometry applications. Previously disclosedmethods using laser ablation of a tissue cross-section can be timeconsuming and as disclosed herein the rate of sample introduction can belimited by spread of the aerosol plasma. In methods provided by thepresent disclosure, a tissue sample can be prepared on the structuredcarrier and stored for later imaging mass cytometry or imaging massspectrometry analysis. Also, prior to IMC analysis, the sample may beassessed using optical, fluorescence, or other microscopy to identifyspecific areas of interest for subsequent IMC analysis.

Sample Desorption

In certain embodiments, the sample carrier is not moved with respect toother elements of the sample introduction apparatus such as with respectto a desorption source or the gas flow. Mechanical movement of thesample carrier such as with a translation stage can reduce the rate atwhich individual pixels can be generated and thereby reduce systemthroughout. As discussed below, desorption can be achieved by a varietyof techniques, including lifting.

The terms desorb and desorption are generally used to refer to releaseof a biological sample for discrete sites on a carrier surface, withoutsubstantial vaporization (e.g., a majority of the sample not beingvaporized). The terms include any suitable method such as thermal,photolytic, chemical, or physical. Desorb and desorption aredistinguished from ablation and other processes in which a sample ofbiological material is substantially vaporized at the time it isreleased from a substrate.

In certain embodiments, a biological sample may be released from thesample by thermal mechanisms. In such embodiments, the surface of thediscrete site becomes sufficiently hot to desorb the biologicalmaterial. Heat can be provided by a radiative source such as a laser.The energy applied to the surface is sufficient to desorb the biologicalmaterial, preferably without altering the biological sample. Anysuitable radiation wavelength can be used, which can depend in part onthe absorptive properties of a surface. In certain embodiments, asurface or layer of a discrete site may be coated with or include anabsorber that absorbs incident radiation for conversion to heat. Theradiation may be delivered to a top surface of a discrete site or to abottom surface of a discrete site through the thickness of the carrier.The heated surface may be a surface layer or may be an inner layer of amultilayer structure of a discrete site. Desorption by heating can takeplace on a nanosecond time scale. Also, vaporization of the supportlayer can occur on a picosecond time scale when a picosecond laserpulses are used.

In certain embodiments, a discrete site can include a layer of anelectrical conductor that heats up upon the application of a current. Insuch cases discrete sites are electrically connected to electrodes andthe discrete sites are individually addressable.

In certain embodiments, a biological sample may be attached to adiscrete site with a cleavable photoreactive moiety. Upon irradiatingthe cleavable photoreactive moiety with a suitable light source, thephotoreactive moiety can cleave to release the biological sample.

In certain embodiments, a discrete site may include a coating or layerof a chemically reactive species that imparts kinetic energy to thebiological sample to release the sample from the surface. For example, achemically reactive species may release a gas such as, for example, H₂,CO₂, N₂ or hydrochlorofluorocarbons. Examples of such compounds includeblowing and foaming agents, which release gas upon heating. Generationof gas can be used to impart kinetic energy to a desorbing biologicalsample that can improve the reproducibility and direction of release ofbiological samples from the discrete sites.

In certain embodiments, a discrete site may have photoinitiated chemicalreactants that undergo an exothermic reaction to generate heat fordesorbing a biological material.

In certain embodiments, the discrete sites may be mounted and/or coupledto MEMS devices configured to facilitate release of a biologicalmaterial from the discrete sites on a carrier.

Optionally, a laser may be focused at a location adjacent to the samplebeing lifted, rather than at the sample location. For example, the lasermay be focused on the order of a few micrometers adjacent to the samplebeing lifted. Lifting ablation will be quite violent (as all ablationsare) but by separating the ablation from the cell, the lifting may begentler. Put in another way, the separation of the laser ablationlocation from the sample location of a few microns (e.g., less than 10microns, less than 5 microns, less than 3 microns, or the like) providesa cushion and the acting media that lifts the cell may be a compressionwave generated by the nearby ablation. The ablation of material adjacentthe sample may lift the sample from the supporting surface and into thegas phase.

In some embodiments, the sample may be supported by a portion of asupport film. The film may be attached to the substrate. A laser may befocused around the edge of the biological sample to separate the portionof the support film from a remainder of the support film. Thereafter,the portion of the support film supporting the sample may be gentlyheated (e.g., using the same or a different laser) to lift the sampleinto the gas phase. This two-step cutting and separation approach mayreduce the amount of energy required to lift the cell and may make thelifting less violent to reduce the chances of damaging the cell duringlifting. For example, in some embodiments, an IR laser may be used tocut around an area of interest while a UV laser may be used to nudge thecell and the film off the substrate (if a substrate is used—sometimesthe sample may just reside on the film).

This may be advantageous when the sample is irregularly or randomlycaptured. In some embodiments, the analysis systems may include lasersteering components to steer the focal point relative to the sample inorder to separate the portion of the support film from a remainder ofthe film. Additionally, as discussed above, the cells can be selectedbased on imaging techniques and/or other criteria. For example, U.S.patent application Ser. No. 14/060,125 describes methods and systems forinterrogating a sample (e.g., fluorescence microscopy) to identifylocations of target cells for location specific analysis that may beused with embodiments described herein. Such methods and systems may beparticularly advantageous when the biological material has an irregularor random arrangement on the sample support.

Single cell analysis by mass cytometry is limited by the introductionrate and is currently much less than in fluorescent flow cytometrysystems. Fluorescent flow cytometers are capable of analyzing around30,000 cells/sec compared to about 1,000 cells/sec for current masscytometers. Limitations associated with spread of the aerosol plume ofthe biological sample in the laser ablation chamber and during transportto the ionization source, for example an ICP source, further limits theability to isolate discrete samples of biological material. Methods havebeen developed for introducing biological material such as dropletscontaining individual cells directly into an ionization source, forexample an ICP source, using microfluidics. Verboket et al., Anal. Chem.2014, 86, 6012-6018. However, such methods appear to be limited tosample introduction rates around 300 cells/sec and these methods do notprevent cases when multiple cells aggregate in one droplet.

Using apparatus and methods provided by the present disclosure it isanticipated that sample introduction rates from 1,000 cells/sec to 5,000cells/sec are possible with the advantage of predictability of timing ofcell arrival as well as removal of the instances when multiple cells arearriving simultaneously.

FIG. 6 shows an example of a sample carrier with discrete sample sitesdisposed on a surface of the carrier. Each of the sample sites includesa capture layer and a desorption film underlying the capture layer. Thebiological material is situated on the capture layer. The capture layermay be configured to capture the biological material in any of themethods described above (e.g., mechanically, chemically,electrostatically, hydrophilic and/or hydrophobic interactions,combinations thereof, or the like). In this embodiment, the carrier istransparent to certain radiation such that for example laser radiationfocused from the backside of the carrier onto the desorption film causesthe sample to be released into the carrier gas flow and into the gasconduit. The desorption film can absorb the radiation to cause releaseof the desorption film and/or the biological sample. In someembodiments, the desorption film is thin. In some embodiments, thedesorption film can be 100 nm thick, or it can be up to 1 micrometerthick. The laser light properties (wavelength, pulse duration) may beselected to provide absorption of a large portion of the laser energyinto the film. The film can be made as thin as possible to minimize theenergy needed for ablation of the film. Some materials and laserparameters might support absorption of the energy into an even thinnerlayers such as 10 nm or 30 nm. The desorption film may be various formsof plastics (such as PEN, PMMA, Kapton, etc.) and may be configured toabsorb energy from UV lasers. Optionally, the desorption film may betriazene polymer or metallic layers. In further embodiments, thedesorption film may be precut such that the desorption film is separatedfrom adjacent desorption films associated with an adjacent pixel. Insome embodiments, the desorption film may be precut prior to samplelifting using a laser—which could be a separate or the same laser thanthe laser used to heat or ablate the desorption layer during samplelifting. The precut desorption film may reduce the amount of energyrequired for lifting the sample into the gas phase and may therebyreduce the possibility of vaporizing or damaging the sample duringlifting (e.g., via laser ablation or heating of the desorption film).Additionally, while not shown, a cushion layer may be disposed betweenthe desorption layer and the capture layer. For instance, a thickerlayer of plastic can be deposited on top of desorption/ablation layer.The cushion layer can be 1 micrometer thick while the ablation layer canbe 100 nm. In this case the velocity and acceleration of the ablationlayer will be reduced (cushioned) by about 10× when it propagates to thebiological material due to inertia of the cushion layer. With thecushion layer it may be desirable to make it such that the area beinglifted easily separates from the bulk or remainder of the cushion layer.Pre-cutting or patterning of the cushion layer can be useful.

The cushion layer may be provided to further protect the biologicalsample during lifting of the biological sample. For example, if thedesorption layer is ablated to eject the biological sample from thesupport, the cushion layer may be configured to absorb the excess energyfrom the ablation to protect the biological sample during ejection.

FIG. 7 shows an alternative configuration in which radiation such aslaser radiation is directed onto the front side of the sample. The laserradiation penetrates the biological sample and is absorbed thedesorption film thereby causing the sample to be released from thecapture site. The properties of the laser radiation are selected so thatthe biological sample remains intact and is not itself vaporized orablated by the laser radiation.

In certain embodiments, a biological sample can be released or desorbedfrom a discrete site using nano-heaters, bubble jets, piezoelectrics,ultrasonics, electrostatics, or a combination of any of the foregoing.

Each, or a combination, of these techniques permits ordered detachmentof sample material from the sample carrier. However, often, the locationon the sample that is of interest does not represent a discrete entity,such as a lone cell, at a discrete site which can be easily lifted inisolation. Instead, the cell of interest may be surrounded by othercells or material, of which analysis is not required or desired. Tryingto perform desorption (e.g. lifting) of the location of interest maytherefore desorb both the cell of interest and surrounding materialtogether. Atoms, such as labelling atoms which are used in elementaltags (see discussion below), from the surrounding area of the sample(e.g. from other cells which have been labelled) that are carried in adesorbed slug of material in addition to the specific location (e.g.cell) of interest could therefore contaminate the reading for thelocation of interest.

A solution to this problem is provided by the invention whereby thetechniques of ablation and desorption (such as by lifting) can becombined in a single method. For example, to perform precise desorptionof a location (e.g. cell) of interest on a biological sample, e.g. atissue section sample or cell suspension dispersion, on the samplecarrier, laser ablation can be used to ablate the area around the cellof interest to clear it of other material. After clearing thesurrounding area by ablation, the location of interest can then bedesorbed from the sample carrier, and then ionized and analyzed by massspectrometry in line with standard mass cytometry or mass spectrometryprocedures. In line with the above discussion, thermal, photolytic,chemical, or physical techniques can be used to desorb material from alocation of interest, optionally after ablation has been used to clearthe area surrounding the location that will be desorbed. Often, liftingwill be employed, to separate the slug of material from the samplecarrier (e.g. a sample carrier which has been coated with a desorptionfilm to assist the lifting procedure, as discussed above with regard todesorption from discrete sites). The slug desorbed from the sample e.g.after clearing, is thus akin to the “pixel” discussed elsewhere hereinwith regard to material desorbed from discrete sites on structuredsample carriers.

FIG. 8 is a schematic of the steps of the combined ablation and liftingmethod. In FIG. 8A, first laser radiation (A) is directed on the sample(B), which ablates that part of the sample (as indicated by the gapbetween sample fragments (C) and the slug of material to be analyzed(D), in FIGS. 8B and 8C). The sample (B) is on a sample carrier (E) andbetween the sample and the carrier is a functionalized layer (F) of thetype discussed in the preceding paragraphs, such as a desorption filmcoating, which assists the desorption of sample material from thesurface of carrier. FIGS. 8B and 8C illustrate the same step—theirradiation of the functionalized layer (F) with laser radiation (G).FIGS. 8B and 8C are alternative modes of irradiation. 8B showsirradiation of the functionalized layer (F) through the sample carrier(E), whereas 4C shows irradiation of the functionalized layer (F)through the sample material to be desorbed (D). FIG. 8D illustrates theproduction of a gas (H) by the functionalized layer (F) followingirradiation which ejects the slug of sample material (D) into the gasphase, wherein it is carried by the flow of gas (I) into the conduit (J)leading to the ionization system. In some instances, there is noproduction of gas by the functionalized layer to eject the slug ofsample material, and instead another kind of laser-induced desorptionoccurs when the functionalized layer, such as a desorption film, absorbslaser radiation.

Accordingly, the invention provides a method of analyzing a samplecomprising:

(i) performing laser ablation of a sample using laser radiation;

(ii) desorbing a slug of sample material using laser radiation; and

(iii) ionizing the slug of sample material and detecting atoms in theslug by mass spectrometry.

In some embodiments, the ablation of the sample generates one or moreplumes of sample material produced, and wherein the plumes areindividually ionized and the atoms in the plume detected by massspectrometry. In some embodiments, the sample is on a sample carriercomprising a functionalized layer, and laser radiation targets thefunctional layer, of the kind discussed above (such as a desorptionfilm), in order to cause the desorption of the slug of sample materialthat separates it from the sample (i.e. lifting).

In some embodiments of the invention, the parts of the sample that areremoved by desorption and by ablation may be different. For example,where there is a cluster of cells, ablation (such as with subcellularresolution) may be performed, to enable the imaging of all cells in thecluster (e.g. where desorbing the sample material could remove multiplecells at once, which may not be desired where cell-by-cell analysis isrequired). On the same sample, however, there may be cells which arespaced apart from the other cells, and so can be lifted. In someembodiments, step (ii) is performed before step (i).

In some embodiments, the sample is a biological sample, such as a tissuesection, or a cell solution dispersed on the slide (and optionallyfixed). In some embodiments, prior to step (i), the method comprises theadditional step of labelling a plurality of different target moleculesin the sample with one or more different labelling atoms, to provide alabelled sample. The labelling step thereby enables imaging masscytometry, in addition to imaging mass spectrometry.

In some embodiments, the laser ablation is used to ablate the materialaround a location of interest to clear the surrounding area before thesample material at the location of interest is desorbed as a slug ofmaterial (e.g. by lifting). This slug of material is then analyzed, e.g.in the same way as the pixels discussed elsewhere herein, generated whenstructured sample carriers are employed.

The location of interest can be identified by another technique beforethe laser ablation and desorption (e.g. by lifting) is performed. Theinclusion of a camera (such as a charged coupled device image sensorbased (CCD) camera or a CMOS camera or an active pixel sensor basedcamera), or any other light detecting means in an imaging massspectrometer as described in the preceding sections is one way ofenabling these techniques. The camera can be used to scan the sample toidentify cells of particular interest or locations of particularinterest (for example cells of a particular morphology). Once suchlocations have been identified, the locations can be lifted after laserpulses have been directed at the area around the location of interest toclear other material by ablation before the location (e.g. cell) islifted. This process may be automated (where the system both identifies,ablates and lifts the location(s) of interest) or semi-automated process(where the user of the system, for example a clinical pathologist,identifies the location(s) of interest, following which the system thenperforms ablation and lifting in an automated fashion). This enables asignificant increase in the speed at which analyses can be conducted,because instead of needing to ablate the entire sample to analyzeparticular cells, the cells of interest can be specifically ablated.

The camera can record the image from a confocal microscope. Theidentification may be by light microscopy, for example by examining cellmorphology or cell size. Sometimes, the sample can be specificallylabelled to identify the location(s) (e.g. cell(s)) of interest.

Often, fluorescent markers are used to specifically stain the cells ofinterest (such as by using labelled antibodies or labelled nucleicacids). These fluorescent makers can be used to stain specific cellpopulations (e.g. expressing certain genes and/or proteins) or specificmorphological features on cells (such as the nucleus, or mitochondria)and when illuminated with an appropriate wavelength of light, theseregions of the sample are specifically identifiable. In some instances,the absence of a particular kind fluorescence from a particular area maybe characteristic. For instance, a first fluorescent label targeted to acell membrane protein may be used to broadly identify cells, but then asecond fluorescent label targeted to the ki67 antigen (encoded by theMKI67 gene) can discriminate between proliferating cells andnon-proliferating cells. Thus by targeting cells which lack fluorescencefrom the second label fluorescent, non-replicating cells can bespecifically targeted for analysis. In some embodiments, the systemsdescribed herein therefore can comprise a laser for excitingfluorophores in the labels used to label the sample. Alternatively, anLED light source can be used for exciting the fluorophores. Non confocal(e.g. wide field) fluorescent microscopy can also be used to identifycertain regions of the biological sample, but with lower resolution thanconfocal microscopy.

When a laser is used to excite fluorophores for fluorescence microscopy,in some embodiments this laser is the same laser that generates thelaser radiation used to ablate material from the biological sample andfor lifting (desorption), but used at a fluence that is not sufficientto cause ablation or desorption of material from the sample. In someembodiments, the fluorophores are excited by a wavelength of laserradiation that is used for sample desorption. The laser radiation thatexcites the fluorophores may be provided by a different laser sourcefrom the ablation and/or lifting laser source(s).

By using an image sensor (such as a CCD detector or an active pixelsensor, e.g. a CMOS sensor), it is possible to entirely automate theprocess of identifying regions of interest and then ablating them, byusing a control module (such as a computer or a programmed chip) whichcorrelates the location of the fluorescence with the x,y coordinates ofthe sample and then directs the ablation laser radiation to the areasurrounding that location before the cell at the location is lifted. Aspart of this process, in some embodiments, the first image taken by theimage sensor has a low objective lens magnification (low numericalaperture), which permits a large area of the sample to be surveyed.Following this, a switch to an objective with a higher magnification canbe used to home in on the particular features of interest that have beendetermined to fluoresce by higher magnification optical imaging. Thesefeatures recorded to fluoresce may then be lifted. Using a lowernumerical aperture lens first has the further advantage that the depthof field is increased, thus meaning features buried within the samplemay be detected with greater sensitivity than screening with a highernumerical aperture lens from the outset.

In methods and systems in which fluorescent imaging is used, theemission path of fluorescent light from the sample to the camera mayinclude one or more lenses and/or one or more optical filters. Byincluding an optical filter adapted to pass a selected spectralbandwidth from one or more of the fluorescent labels, the system isadapted to handle chromatic aberrations associated with emissions fromthe fluorescent labels. Chromatic aberrations are the result of thefailure of lenses to focus light of different wavelengths to the samefocal point. Accordingly, by including an optical filter, the backgroundin the optical system is reduced, and the resulting optical image is ofhigher resolution. A further way to minimize the amount of emitted lightof undesired wavelengths that reaches the camera is to exploit chromaticaberration of lenses deliberately by using a series of lenses designedfor the transmission and focus of light at the wavelength transmitted bythe optical filter, akin to the system explained in WO 2005/121864.

A higher resolution optical image is advantageous in this coupling ofoptical techniques and lifting, because the accuracy of the opticalimage then determines the precision with which the ablating laser sourcecan be directed to ablate the area surrounding the cell of interest.

Accordingly, the invention provides a method of performing masscytometry on a sample comprising a plurality of cells, the methodcomprising steps of: (i) labelling a plurality of different targetmolecules in the sample with one or more different labelling atoms, toprovide a labelled sample; (ii) illuminating the sample with light toidentify one or more locations of interest; (iii) recording locationalinformation of the one or more locations of interest on the sample; (iv)using the locational information of the locations of interest to desorba slug of sample material from a location of interest, comprises firstperforming laser ablation to remove sample material surrounding thelocation of interest, before the slug of sample material is desorbedfrom the location using laser radiation; (v) ionizing the desorbed slugof sample material; and (vi) subjecting the ionised sample material tomass spectrometry, for detection of labelling atoms in the samplematerial.

The invention also provides a method of performing mass cytometry on asample comprising a plurality of cells, the method comprising steps of:(i) labelling a plurality of different target molecules in the samplewith one or more different labelling atoms and one or more fluorescentlabels, to provide a labelled sample; (ii) illuminating the sample withlight to excite the one or more fluorescent labels; (iii) recordinglocational information of one or more locations of the sample based onthe pattern of fluorescence; (iv) using the locational information basedon the pattern of fluorescence to desorb a slug of sample material froma location of interest, comprising first performing laser ablation toremove sample material surrounding the location of interest, before theslug of sample material is desorbed from the location; (v) ionizing thedesorbed slug of sample material; and (vi) subjecting the ionised samplematerial to mass spectrometry, for detection of labelling atoms in thesample material.

In some embodiments of the methods described above, the sample is on asample carrier (e.g. microscope slide or other appropriate solidplatform from which the sample can be ablated or desorbed, typicallywithout significant removal of atoms of the carrier). In someembodiments, the laser radiation used to desorb sample material isdirected to the sample material through the sample carrier. In someembodiments, the slug of sample material is desorbed by lifting. In someembodiments, the sample is on a sample carrier which comprises a layerwhich absorbs laser radiation to assist lifting of the slug of samplematerial. Examples of suitable layers, which may be a desorption filmcoated on the surface of the sample carrier, include triazene polymer,such as shown in FIG. 1 of Doraiswamy et al., 2006, Applied SurfaceScience, 52: 4743-4747, or other polymers which evaporate on upon laserradiation.

In some embodiments, no data are recorded from the ablation performed toclear the area around the location to be desorbed (e.g. the cell ofinterest). In some embodiments, data is recorded from the ablation ofthe surrounding area. Useful information that can be obtained from thesurrounding area includes what target molecules, such as proteins andRNA transcripts, are present in the surrounding cells and intercellularmilieu. This may be of particular interest when imaging solid tissuesamples, where direct cell-cell interactions are common, and whatproteins etc. are expressed in the surrounding cells may be informativeon the state of the cell of interest.

The invention also provides an imaging mass spectrometer or imaging masscytometer, comprising a control module programmed to perform the methodsset out in this section.

Gas Conduit

Following desorption (e.g. by lifting) of a biological sample from thecarrier, the biological sample can be injected into a gas stream andsubsequently introduced into an ionization source, for example an ICPtorch, where the biological sample is ionized and the ions subsequentlyrecorded by suitable mass spectrometry methods.

The carrier gas may be any suitable gas for use in mass cytometry ormass spectrometry including inert gases such as argon, helium, or acombination thereof.

The carrier gas can flow across the surface of a carrier and confined atone end into a narrow flow tube or channel. Much like a fluidic flowcytometer, the carrier gas and entrained biological sample can be gasdynamically focused such that individual pixels enter the ionizationsource, for example an ICP torch, one at a time.

The length of the gas conduit can be any suitable length with theobjective of delivering desorbed biological sample from the carrier tothe ionization source, for example an ICP source, of a mass cytometer ormass spectrometer. Based largely on practical considerations the lengthof the gas conduit can be from about 10 cm to about 40 cm.

The diameter of the gas conduit can be any suitable diameter, again withthe objective of delivering desorbed biological sample from the carrierto the ionization source, for example an ICP source, of a mass cytometeror mass spectrometer. For example, in certain embodiments a gas conduitcan have an inner diameter from about 0.2 mm to about 3 mm. It isdesirable that the inner diameter of the gas conduit be sufficientlylarge to minimize clogging. The conduit can have a certain diameterthroughout the most of its length but can also taper down to a smallerdiameter near the ICP torch as commonly practiced for ICP torchinjectors. Again, the dynamics along the gas conduit is established andmanipulated to facilitate a continuous stream of discrete materialpixels into the ICP source. Ideally the pixels will be located towardthe center of the gas conduit where the gas velocity is a maximum andthe change in velocity with position is minimized. In certainembodiments, the carrier gas flow through the conduit is characterizedby laminar gas flow with a parabolic profile center along the axis ofthe gas conduit.

Mass Cytometry Analysis

In certain embodiments, a biological sample is labeled with an elementtag. Element tags comprising metal polymer conjugates can provide highlymultiplexed sensitive assays for biological material; the metal atomsare also termed labelling atoms. The element tags can be conjugated toaffinity molecules such as antibodies, which can be used forquantitative proteomics and genomics of biological samples. Element tagsare described, for example in U.S. Application No. 2008/0003616, whichis incorporated by reference in its entirety. Analysis of biologicalmaterial using element tags is described, for example, in U.S.Application Publication Nos. 2010/0144056, 2012/0061561, 2014/0120550,and 2014/0121117, and WO 2014/169294, each of which is incorporated byreference in its entirety. In general, the materials and methodsdisclosed in these publications can be applied to the embodimentsprovided by the present disclosure, with the primary difference beingthat in the publications, the biological material is ablated orvaporized and transferred as a vapor plume from the sample carrier tothe ionization source, for example an ICP source. In contrast, in theembodiments provided by the present disclosure, the biological materialis desorbed (e.g., lifted, released, or ejected) from the carriersurface as an un-vaporized material pixel, which becomes entrained in acarrier gas flow to bring the material sample to the ionization source,for example an ICP source.

Labelling of the Biological Sample

In some embodiments, as described above, the apparatus and methods ofthe invention detect atoms that have been added to a sample (i.e. whichare not normally present). As noted above such atoms are calledlabelling atoms. The sample is typically a biological sample comprisingcells, and the labelling atoms are used to label target molecules in thecells/on the cell surface. In some embodiments, simultaneous detectionof many more than one labelling atom, permitting multiplex labeldetection e.g. at least 3, 4, 5, 10, 20, 30, 32, 40, 50 or even 100different labelling atoms is enabled. Labelling atoms can also be usedin a combinatorial manner to even further increase the number ofdistinguishable labels. By labelling different targets with differentlabelling atoms it is possible to determine the presence of multipletargets on a single cell.

Labelling atoms that can be used with the invention include any speciesthat are detectable by MS and that are substantially absent from theunlabelled sample. Thus, for instance, ¹²C atoms (carbon 12) would beunsuitable as labelling atoms because they are naturally abundant,whereas ¹¹C could in theory be used because it is an artificial isotopewhich does not occur naturally. In preferred embodiments, however, thelabelling atoms are transition metals, such as the rare earth metals(the 15 lanthanides, plus scandium and yttrium). These 17 elementsprovide many different isotopes which can be easily distinguished by MS.A wide variety of these elements are available in the form of enrichedisotopes e.g. samarium has 6 stable isotopes, and neodymium has 7 stableisotopes, all of which are available in enriched form. The 15 lanthanideelements provide at least 37 isotopes that have non-redundantly uniquemasses. Examples of elements that are suitable for use as labellingatoms include Lanthanum (La), Cerium (Ce), Praseodymium (Pr), Neodymium(Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium, (Gd),Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm),Ytterbium (Yb), Lutetium (Lu), Scandium (Sc), and Yttrium (Y). Inaddition to rare earth metals, other metal atoms are suitable fordetection by MS e.g. gold (Au), platinum (Pt), iridium (Ir), rhodium(Rh), bismuth (Bi), etc. The use of radioactive isotopes is notpreferred as they are less convenient to handle and are unstable e.g. Pmis not a preferred labelling atom among the lanthanides.

In order to facilitate time of flight analysis it is helpful to uselabelling atoms with an atomic mass within the range 80-250 e.g. withinthe range 80-210, or within the range 100-200. This range includes allof the lanthanides, but excludes Sc and Y. The range of 100-200 permitsa theoretical 101-plex analysis by using different labelling atoms,while permitting the invention to take advantage of the high spectralscan rate of TOF MS. As mentioned above, by choosing labelling atomswhose masses lie in a window above those seen in an unlabelled sample(e.g. within the range of 100-200), TOF detection can be used to providerapid analyses at biologically significant levels.

Labelling the particles generally requires that the labelling atoms areattached to one member of a specific binding pair (sbp). This labelledsbp is contacted with a sample such that it can interact with the othermember of the sbp (the target sbp member) if it is present, therebylocalizing the labelling atom to a target molecule in the sample. Themethod of the invention then detects the presence of the labelling atomon a particle as it is analyzed by the mass cytometer. Rare earth metalsand other labelling atoms can be conjugated to sbp members by knowntechniques e.g. Bruckner et al. (2013) Anal. Chem. 86:585-91 describesthe attachment of lanthanide atoms to oligonucleotide probes for MSdetection, Gao & Yu (2007) Biosensor Bioelectronics 22:933-40 describesthe use of ruthenium to label oligonucleotides, and Fluidigm Canadasells the MaxPar™ metal labelling kits which can be used to conjugateover 30 different labelling atoms to proteins (including antibodies).

Various numbers of labelling atoms can be attached to a single sbpmember, and greater sensitivity can be achieved when more labellingatoms are attached to any sbp member. For example greater than 10, 20,30, 40, 50, 60, 70, 80, 90 or 100 labelling atoms can be attached to asbp member. For example, monodisperse polymers containing multiplemonomer units may be used, each containing a chelator such as DTPA.DTPA, for example, binds 3+ lanthanide ions with a dissociation constantof about 10⁻⁶ M. These polymers can terminate in a thiol-reactive group(e.g. maleimide) which can be used for attaching to a sbp member. Forexample the thiol-reactive group may bind to the Fc region of anantibody. Other functional groups can also be used for conjugation ofthese polymers e.g. amine-reactive groups such as N-hydroxy succinimideesters, or groups reactive against carboxyls or against an antibody'sglycosylation. Any number of polymers may bind to each sbp member.Specific examples of polymers that may be used include straight-chain(“X8”) polymers or third-generation dendritic (“DN3”) polymers, bothavailable as MaxPar™ reagents. Use of metal nanoparticles can also beused to increase the number of atoms in a label.

As mentioned above, labelling atoms are attached to a sbp member, andthis labelled sbp member is contacted with the sample where it can findthe target sbp member (if present), thereby forming a labelled sbp. Thelabelled sbp member can comprise any chemical structure that is suitablefor attaching to a labelling atom and then for detection according tothe invention.

In general terms, methods of the invention can be based on any sbp whichis already known for use in determining the presence of target moleculesin samples (e.g. as used in IHC or fluorescence in situ hybridisation,FISH) or fluorescence-based flow cytometry, but the sbp member which iscontacted with the sample will carry a labelling atom which isdetectable by MS. Thus the invention can readily be implemented by usingavailable flow cytometry reagents, merely by modifying the labels whichhave previously been used e.g. to modify a FISH probe to carry a labelwhich can be detected by MS.

The sbp may comprise any of the following: a nucleic acid duplex; anantibody/antigen complex; a receptor/ligand pair; or an aptamer/targetpair. Thus a labelling atom can be attached to a nucleic acid probewhich is then contacted with a sample so that the probe can hybridize tocomplementary nucleic acid(s) therein e.g. to form a DNA/DNA duplex, aDNA/RNA duplex, or a RNA/RNA duplex. Similarly, a labelling atom can beattached to an antibody which is then contacted with a sample so that itcan bind to its antigen. A labelling atom can be attached to a ligandwhich is then contacted with a sample so that it can bind to itsreceptor. A labelling atom can be attached to an aptamer ligand which isthen contacted with a sample so that it can bind to its target. Thuslabelled sbp members can be used to detect a variety of target moleculesin a sample, including DNA sequences, RNA sequences, proteins, sugars,lipids, or metabolites.

In a typical embodiment of the invention the labelled sbp member is anantibody. Labelling of the antibody can be achieved through conjugationof one or more labelling atom binding molecules to the antibody, forexample using the MaxPar™ conjugation kit as described above. The targetmolecule of an antibody is called its antigen, and may be a protein,carbohydrate, nucleic acid etc. Antibodies which recognize cellularproteins that are useful for mass cytometry are already widely availablefor IHC usage, and by using labelling atoms instead of current labellingtechniques (e.g. fluorescence) these known antibodies can be readilyadapted for use in methods of the invention, but with the benefit ofincreasing multiplexing capability. Antibodies used with the inventioncan recognize targets on the cell surface or targets within a cell.Antibodies can recognize a variety of targets e.g. they can specificallyrecognize individual proteins, or can recognize multiple relatedproteins which share common epitopes, or can recognize specificpost-translational modifications on proteins (e.g. to distinguishbetween tyrosine and phospho-tyrosine on a protein of interest, todistinguish between lysine and acetyl-lysine, to detect ubiquitination,etc.). After binding to its target, labelling atom(s) conjugated to anantibody can be detected to reveal the presence of that target in asample.

The labelled sbp member will usually interact directly with a target sbpmember in the sample. In some embodiments, however, it is possible forthe labelled sbp member to interact with a target sbp member indirectlye.g. a primary antibody may bind to the target sbp member, and alabelled secondary antibody can then bind to the primary antibody, inthe manner of a sandwich assay. Usually, however, the invention relieson direct interactions, as this can be achieved more easily and permitshigher multiplexing. In both cases, however, a sample is contacted witha sbp member which can bind to a target sbp member in the sample, and ata later stage label attached to the target sbp member is detected.

One feature of the invention is its ability to detect multiple (e.g. 10or more, and even up to 100 or more) different target sbp members in asample e.g. to detect multiple different proteins and/or multipledifferent nucleic acid sequences on particles such as cells or beads. Topermit differential detection of these target sbp members theirrespective sbp members should carry different labelling atoms such thattheir signals can be distinguished by MS. For instance, where tendifferent proteins are being detected, ten different antibodies (eachspecific for a different target protein) can be used, each of whichcarries a unique label, such that signals from the different antibodiescan be distinguished. In some embodiments, it is desirable to usemultiple different antibodies against a single target e.g. whichrecognize different epitopes on the same protein. Thus a method may usemore antibodies than targets due to redundancy of this type. In general,however, the invention will use a plurality of different labelling atomsto detect a plurality of different targets.

If more than one labelled antibody is used with the invention, it ispreferable that the antibodies should have similar affinities for theirrespective antigens, as this helps to ensure that the relationshipbetween the quantity of labelling atoms detected by MS and the abundanceof the target antigen will be more consistent across different sbps(particularly at high scanning frequencies).

If a target sbp member is located intracellularly, it will typically benecessary to permeabilise cell membranes before or during contacting ofthe sample with the labels. For example when the target is a DNAsequence but the labelled sbp member cannot penetrate the membranes oflive cells, the cells of the sample can be fixed and permeabilised. Thelabelled sbp member can then enter the cell and form a sbp with thetarget sbp member.

Usually, a method of the invention will detect at least oneintracellular target and at least one cell surface target. In someembodiments, however, the invention can be used to detect a plurality ofcell surface targets while ignoring intracellular targets. Overall, thechoice of targets will be determined by the information which is desiredfrom the method.

Accordingly, in some embodiments, the methods of analysis describedabove comprise the step of labelling a sample with at least onelabelling atom. This atom can then be detected using the methodsdescribed above.

Finally, it should be noted that there are alternative ways ofimplementing the embodiments disclosed herein. Accordingly, the presentembodiments are to be considered as illustrative and not restrictive.Furthermore, the claims are not to be limited to the details givenherein, and are entitled their full scope and equivalents thereof.

1.-37. (canceled)
 38. A method of delivering a biological sample into amass cytometry system, the method comprising: focusing a first laserspot onto a film supporting the biological sample to heat the film,wherein heating of the film lifts the biological sample into a gas phasewithout complete vaporization of the biological sample, the gas phaseconfigured to deliver the biological sample to the mass cytometrysystem.
 39. The method of claim 38, wherein the film is coupled with asubstrate, and wherein the film comprises a plurality of separateportions of film at discrete spaced apart sites on the substrate. 40.The method of claim 39, wherein the plurality of separate portions offilm define a plurality of cell capture sites, wherein the cell capturesites are configured to capture only a single cell.
 41. The method ofclaim 39, wherein a surface of gaps between the plurality of separateportions of film are modified to repel the biological sample.
 42. Themethod of claim 38, wherein the first laser spot is focused onto thefilm supporting the biological sample to ablate the film.
 43. The methodof claim 42, wherein a cushion layer is disposed between the biologicalsample and the film, the cushion layer configured to absorb energy fromthe ablation of the film to limit damage to the biological sample fromthe ablation of the film.
 44. The method of claim 43, wherein a surfaceof the cushion layer is modified to preferentially capture a targetbiological sample.
 45. The method of claim 42, further comprisingfocusing a cutting laser spot onto the film to cut around the biologicalsample to separate the portion of the film supporting the biologicalsample from a bulk of the film.
 46. The method of claim 45, wherein thecutting laser spot is from a first laser, and wherein the first laserspot is from a second laser different from the first laser.
 47. Themethod of claim 38, further comprising flowing a carrier gas over thebiological sample from a gas conduit, and translating the biologicalsample relative to the gas conduit.
 48. The method of claim 38, whereinthe biological sample is applied to a sample carrier, and wherein thesample carrier includes a corrugated surface.
 49. The method of claim48, wherein the corrugated surface of the sample carrier includes raisededges configured to cutting a tissue section into one or more individualregions.
 50. A method of analyzing a sample comprising: (i) performinglaser ablation of a sample using laser radiation; (ii) desorbing a slugof sample material; and (iii) ionizing the slug of sample material anddetecting atoms in the slug by mass spectrometry.
 51. The method ofclaim 50, in which laser ablation is used to ablate the material arounda location of interest to clear the surrounding area before the samplematerial at the location of interest is desorbed from the sample carrieras a slug of material.
 52. The method of claim 50, wherein the sample ison a sample carrier.
 53. The method of claim 52, wherein laser radiationis directed through the sample carrier to desorb the slug of samplematerial from the sample carrier.
 54. A carrier comprising: a surface; aplurality of discrete sites disposed on the surface, wherein, each ofthe plurality of discrete sites is configured to retain a biologicalsample; and each of the plurality of discrete sites is configured torelease the biological sample upon application of an energy pulse. 55.The carrier of claim 54, comprising, a channel underlying the pluralityof discrete sites, wherein the channel extends through a thickness ofthe carrier; and the plurality of discrete sites comprises a holefluidly coupled to the channel.
 56. The carrier of claim 54, wherein thesurface comprises topographic features configured to cut a biologicaltissue, wherein the topographic features are disposed between theplurality of discrete sites.
 57. A method of preparing a structuredbiological sample, comprising: providing the carrier of claim 54,applying a biological sample to the surface of the carrier; andsectioning the biological sample to remove portions of the biologicalsample between the plurality of discrete sites to provide a structuredbiological sample.